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Breakingspecia-onbarriersthroughrandomandCRISPR/Cas9-directedmutagenesisinplants
Rubén Alcázar PLANT STRESS TOLERANCE LABORATORY
Department of Biology, Healthcare & Environment Facultat de
Farmàcia i Ciències de l’Alimentació
Universitat de Barcelona
www.ub.edu/portal/web/dp-bsma/stress
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B1/
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Ler
Kas-2
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SalicylicacidCathecolNahG
EDS1:enhanceddiseasesuscep-bility1SID2:isochorismatesynthase1
RPP1
EDS1
SA
celldeath
Ler/Kas-2incompa)bilityrequiresEDS1andsalicylicacid(SA)competence
expressionchanges
incompa)blelineinEDS1loss-of-func)onmutantincompa)bleline
incompa)blelinetransformedwithNahG
incompa)blelineinSID2loss-of-func)onmutant
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1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
RPP1
-likelocus
Ler Col-0
Col-0RPP1-likeLerRn
RPP1
-likelocus
1 1
2 2
3 3
4 4
5 5
7 7
8 8
At3g44630
At3g44670
TransgenicCol-0linesindividuallytransformedwithRPP1-likeLerR1toR8genes.
Recons)tu)onofincompa)bility.Usinganeutralbackground.
n=1 n=2 n=3 n=4 n=5 n=7 n=8
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R3overexpression(>3-fold)inducesincompa)blephenotypes
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OverexpressionofotherRPP1-likegenesinCol-0doesnotinduceincompa)blephenotypes.
0 4.0-fold
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R3overexpressorlinesareresistanttoHyaloperonosporaarabidopsidis(Noco)
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...butR3isnotsufficienttotriggerincompa)bilitywithKas-2whenexpressionisatwild-typelevels
Co-ac)onoftwoormoreRPP1-likeLergenesisrequiredforincompa)bility.
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R1 R2 R3 R4 R5 6R7 R8620 680
Ler sulki2-1 sulki1-1/10
ScreenforsuppressorsofLer/Kas-2incompa)bility(sulki)mutants
RPP1-likelocus
TIR LRR
G/A R821H
C/T P466L
G/A W484*
C/T S758F
C/T P428S
G/A G877E
GC/CT G509A
G/A G500E
G/A G202E
sulki1-2
sulki1-3
sulki1-4
sulki1-5
sulki1-6
sulki1-8
sulki1-9
sulki1-10
sulki1-1TAAGTAGT/
deletion
sulki1-7
NBRPP1-like Ler R8
TIR LRR
T78I sulki2-1
NBRPP1-like Ler R3
Muta)onsininvariableorhighlyconservedresidues
Notonlythephenotypeisrestored,butalsothetranscrip)onal(RNA-seq)andprimarymetabolism(GC/MS)changesinducedbythehybridincompa-bility.
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Mainconclusions
Specia-oneventscanbetriggeredbysinglenucleo)demuta)ons.Thehybridsexhibitfitnesslossassociatedwithcons)tu)veac)va)onofdefense.Suppressionofdefenserescuescompa)bility,butinducessuscep)bility.Throughrandomordirected(whengenesareknown)mutagenesis,ar-ficially-inducedmuta-onscanbeselectedthatrestorecompa)bilityatnocostondefense.Thisshouldenablethedevelopmentofnewvarie)esforwhichspecia-on(triggeredbyD-Mincompa-bili-es)isabarrier.
